Diversiteit van progenitorcellen in de telencephalon van de Afrikaanse turquoise killivis: van versnelde groei tot verouderen

The African Turquoise killifish (Nothobranchius furzeri) is a promising model for age-related research, offering several advantages over traditional, long-lived vertebrate animal models. Its compressed lifespan allows researchers to study the full spectrum of aging, from early life to death, in a short timeframe. In addition to its rapid growth, the killifish exhibits an age-related decline in regenerative capacity and spontaneous neuronal degeneration, making it an ideal model for studying neurodegenerative diseases associated with aging. Before using the African turquoise killifish to investigate brain aging and related diseases, detailed knowledge of the killifish central nervous system, as well as advanced tools to study them, are required. My PhD research therefore focused on (1) identifying and characterizing the cellular building blocks of the killifish telencephalon at various life stages and (2) the development of transgenic tools to improve the traceability of progenitor cells. This work aimed to elevate the killifish as a model for neural stem cell and regenerative research while enhancing its translational potential as a neurological model. At the start of my PhD, little was known about the cellular diversity of the killifish telencephalon. Single-cell RNA sequencing revealed an extensive diversity of glial and non-glial progenitors, together with other common neural cell types including microglia, oligodendrocytes and a range of neuronal clusters. We identified four distinct radial glia (RG) subtypes, various intermediate cell types, and two atypical non-glial progenitors (NGPs). Spatial mapping further revealed a distinct distribution of these cell clusters in the telencephalon. These spatial maps, combined with the molecular profiles of the four RG subtypes, allowed us to refine their cell identities. We could distinguish two spatially distinct astroglia-like clusters, an ependymal cell cluster in the tela choroidea, and a neuroepithelial-like RG cluster. To characterize the neurogenic niches of the killifish telencephalon, I colocalized cell-specific marker genes with proliferation markers. This demonstrated that the NGP clusters drive the bulk of proliferation in the young-adult killifish, and play critical roles in both neurogenic and gliogenic lineages, contributing to the continuous growth of the killifish telencephalon. In combination with its short lifespan, the killifish undergoes a period of accelerated growth after hatching. Since previous killifish research has primarily focused on aging, the adaptations required for this early-live accelerated growth may have been overlooked. Building on our knowledge of the young-adult and aged killifish, I mapped the cellular architecture during early post-embryonic development and investigated the growth dynamics during this phase. Spatial data revealed key differences between pallial and subpallial regions in terms of growth rate and cellular output. This included a maturing scaffold of radial glia nature already present early post-hatching and a distinct pallial-subpallial patterning of progenitor cells committed to either the glutamatergic or GABAergic neuronal lineage. Collectively, these findings highlight a dynamic and distinct cellular organization for neurogenic regions from pallial and subpallial origin in juvenile stages. To tackle complex questions on neural stem cell function and lineage relationships, the killifish community needs a robust and adaptable genetic toolbox. During my PhD, I optimized killifish-egg injection and in-vivo electroporation techniques to introduce DNA and RNA molecules into the one-cell stage or adult brain, respectively. I used Tol2 transposase and CRISPR-Cas9 techniques to stably integrate long DNA sequences into the killifish genome, contributing to the expanding portfolio of transgenic techniques in the African Turquoise killifish. Taken together, the current transgenic toolbox will advance killifish research significantly. Cell reporter lines will facilitate the study of cell morphology, lineage relationships and the potential of killifish progenitor cells throughout development and aging. Additionally, the development of genetic disease models, including Parkinson's disease and Amyotrophic lateral sclerosis (ALS), will be possible, benefitting the study of disease mechanisms and the identification of novel therapeutic strategies.